DESCRIPTION: The 22 nucleotide let-7 RNA gene is a founding member of a large family of newly identified non-coding RNA genes, called microRNAs (miRNAs), which appear to be present in all multicellular organisms. The let-7 RNA gene was identified by genetic studies in the nematode Caenorhabditis elegans as an important regulator of developmental timing. The let-7 RNA sequence, proteins involved in its expression, including players in the RNA interference (RNAi) pathway, and genes controlled by this RNA are broadly conserved across animal phylogeny. The long-term goal of this proposal is to elucidate how the let-7 RNA is expressed and functions in C. elegans with the expectation that this insight will help reveal the role of let-7 in human health and development. Additionally, this research will contribute to the general understanding of the biogenesis and purpose of the populous family of newly discovered miRNA genes. To test the hypothesis that expression and function of the let-7 RNA gene requires specific RNA sequences and protein factors to produce this regulator of development, the following specific aims will be pursued: 1. Characterize the sequence of let-7 RNA transcripts that give rise to the 22nt product. (A) The sequence of the let-7 RNA primary transcript and processing intermediates will be determined. (B) The let-7 RNA sequence elements that regulate processing will be defined by coupling in vitro assays to in vivo functional analyses. 2. Identify genes involved in the expression of let-7 RNA. (A) Mutations isolated in a forward genetic screen aimed at identifying genes that function with dcr-1, the RNase that processes the approximate 70nt pre-let-7 RNA to the 22nt form, will be characterized and identified. (B) Candidate genes containing ribonuclease, RNA helicase or RNA binding domains will be tested for roles in let-7 RNA maturation by performing processing reactions in extracts where specific genes have been inactivated by RNAi. 3. Determine the factors that mediate let-7-dependent regulation of specific targets. (A) Genes that function with let-7 RNA will be uncovered using a sensitive reporter that is regulated by let-7. (B) The association of factors with the lin-41 3'UTR sequences regulated by let-7 will be characterized using in vitro synthesized RNA probes and C. elegans extracts.